The present invention concerns a deoxyribonucleic acid (DNA) comprising at least one promoter sequence, which is derived from a wild type promoter of a methylotrophic yeast, whose transcription efficiency is modulated compared to the efficiency of the wild type promoter. Further, the present invention concerns host cells, expression vectors, kits and processes for the production of proteins with the use of the DNA according to the invention and various applications thereof and a process for the production of expression vectors.
Because of their capability for mammal-like co- and posttranslational modification of proteins, fungi and in particular yeasts are attractive systems for the production of recombinant proteins. For the production of recombinant proteins, the coding sequence of a gene for a protein of interest is often expressed under the control of a suitable heterologous promoter. The inducible promoters from methylotrophic yeasts have proved especially advantageous here.
Methylotrophic yeasts include for example the genera Candida, Hansenula, Pichia and Torulopsis. Their promoters are characterised by unusually strong transcription induction and good and simple controllability. Particularly advantageous promoters are the promoters responsible in vivo for the regulation of the methanol metabolism. Methylotrophic yeasts catalyse the oxidation of methanol to carbon dioxide via the intermediate stages of formaldehyde and formate, these reactions being catalysed respectively by an alcohol or methanol oxidase (Aoxp or Moxp), a formaldehyde dehydrogenase (Fmdp) and a formate dehydrogenase (Fmdhp). The hydrogen peroxide arising in the first step is degraded by catalase. The C1 compound is assimilated via the transketolase reaction of xylulose-5(P) with formaldehyde. This reaction is catalysed by dihydroxyacetone synthase (Dhasp). The enzymes Aoxp or Moxp, Fmdhp and Dhasp expressed under the control of the AOX or MOX, FMD and DHAS promoters represent up to 40% of the total cell protein in methanol-induced cells. These promoters are thus also described in specialist circles as superpromoters. Most of the promoters for the said genes are repressible with glucose and are therefore very controllable individually.
A further very strong promoter from methylotrophic yeasts is the TPS1 promoter, which controls the expression of the trehalose-6-phosphate synthase gene and is heat-inducible. Tps1p catalyses the conversion of glucose-6-phosphate (GLU6P) and UDP-glucose (UDPG) to trehalose-6-phosphate and UDP. During a one-hour thermal shock from 27° C. to 40° C., treated yeast cells accumulate trehalose and in this way build up increased heat tolerance.
In addition, methylotrophic yeasts have unusually strong constitutive promoters. Two of these very strong constitutive promoters are GAPDH (glyceraldehyde phosphate dehydrogenase) and PMA1 (plasma membrane-bound H+-ATPase 1).
In the recombinant preparation of heterologous proteins, both the activity of the promoter sequence, its regulation and also its integration into the genome of a suitable host cell determine the economic viability and efficiency of the process. In general, especially high transcription activity is desirable. In many cases, however, excessively high transcription activity can lead to the host cell being damaged or dying, before all heterologous gene products of the co- and post-translational modification necessary for the activity could be introduced. In particular, the quantity of toxic proteins must be controlled in such a way that the toxic action is minimised until the optimal yield of transcribed and post-translationally modified proteins. Such control is also difficult to effect with the known promoters inducible with methanol.